Korean Journal of Head & Neck Oncology (대한두경부종양학회지)

The Korean Society for Head and Neck Oncology (대한두경부종양학회)
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Induction of Apoptosis in FRTL-5 Thyroid Cells by Okadaic Acid

Okadaic Acid에 의한 FRTL-5 갑상선 세포주의 Apoptosis 유도

  • Cho Ji-Hyoung (Department of Surgery, Keimyung University College of Medicine) ;
  • Chung Ki-Yong (Department of Surgery, Keimyung University College of Medicine) ;
  • Park Jong-Wook (Department of Immunology, Keimyung University College of Medicine)
  • 조지형 (계명대학교 의과대학 외과학교실) ;
  • 정기용 (계명대학교 의과대학 외과학교실) ;
  • 박종욱 (계명대학교 의과대학 면역학교실)
  • Published : 2002.11.01
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Abstract

Objectve : Okadaic acid is a specific inhibitor of serine/threonine protein phosphatase 1 and 2A. In order to know the mechanism of apoptosis induced by okadaic acid, we treated FRTL-5 thyroid cells with okadaic acid and measured the changes of important proteins that are involved in apoptosis. Materials and Methods: We measured caspase 3 activity, $PLC-{\gamma}1$ degradation, the expression of XIAP, cIAP1, cIAP2, and cytochrome c release in okadaic acid-treated FRTL-5 thyroid cells. Results: Okadaic acid-induced caspase 3 activation and $PLC-{\gamma}1$ degradation and apoptosis were dose-dependent with a maximal effect at a concentration of 80 nmol and time-dependent with a maximal effect at 24 hours after treatment. The elevated caspase 3 activity in okadaic acid treated FRTL-5 thyroid cells are correlated with down-regulation of XIAP and cIAP1, but not cIAP2. General and potent inhibitor of caspases, z-VAD-fmk. abolished okadaic acid-induced caspase 3 activity and $PLC-{\gamma}1$ degradation. The release of cytochrome c in okadaic acid-induced FRTL-5 thyroid cells was dose-dependent with a maximal effect at a concentration of 80 nmol. Conclusions: These findings suggest that mechanism of okadaic acid-induced apoptosis is associated with cytochrome c release and increase of caspase 3 activation in FRTL-5 thyroid cells.

본 연구에서는 FRTL-5 갑상선세포에서 Protein phosphatase type 1(PP-1)과 type 2A(PP-2A) 효소의 억제제인 okadaic acid에 의한 apoptosis 유도 기전에 대하여 이해하고자 하였다. FRTL-5 갑상선세포에 다양한 농도($10{\sim}80nmol$)의 okadaic acid를 처리 한 후 caspase 3와 $PLC-{\gamma}1$ 분절을 확인 한 결과 40nmol 농도에서부터 caspase 3활성과 $PLC-{\gamma}1$ 분절이 나타남을 확인하였다. Okadaic acid에 의한 apoptosis 유도 기전을 조사한 결과 anti-apoptotic 기능이 있는 Bcl-2 단백질 발현은 미약하게 감소하였으나 Bcl-xL은 농도 의존적으로 급격한 감소를 보였다. Caspase 3 효소활성을 저해하는 IAP 단백질 중 cIAP2는 okadaic acid에 영향을 받지 않았으나 cIAP1과 XIAP 단백질 발현은 농도 의존적으로 감소하였다. Okadaic acid에 의한 apoptosis 유도는 caspase 3 의존적인 기전을 보였다. Caspase 3 특이억제제를 okadaic acid와 동시에 처리 시 apoptosis가 억제되었으며 $PLC-{\gamma}1$ 단백질의 절단 현상도 방지되었다. Caspase 3의 활성을 유도하는 cytochrome c의 유리는 okadaic acid처리 농도에 의존적으로 유리하였다. 결론적으로, okadaic acid에 의한 apoptosis 유도는 caspase 3 의존적이며, Bcl-2와 IAP family 단백질 감소현상에 의하여 야기되는 것으로 생각된다.

Keywords

References

  1. Pal S, Choudhuri T, Chattopadhyay S, et al : Mechanisms of curcumin-induced apoptosis of Ehrlichs ascites carcinoma cells. Biochem Biophys Res Commun. 2001 ; 288 : 658-665 https://doi.org/10.1006/bbrc.2001.5823
  2. Gupta S, Mukhtar H : Chemoprevention of Skin Cancer through Natural Agents. Skin Pharmacol Appl Skin Physiol 2001 ; 14 : 373-385 https://doi.org/10.1159/000056371
  3. Lippman SM, Spitz MR : Lung cancer chemoprevention : an integratedapproach. J Clin Oncol. 2001 ; 19 : 74S-82S
  4. Kelloff GJ, Johnson JR, Crowell JA, et al : Approaches to the development and marketing approval of drugs that prevent cancer. Cancer Epidemiol Biomarkers Prev. 1995 ; 4 : 1-10
  5. Ames BN, Gold LS, Willett WC : The causes and prevention of cancer. Proc Natl Acad Sci USA. 1995 ; 92 : 5258-5265 https://doi.org/10.1073/pnas.92.12.5258
  6. Fujiki H, Suganuma M : Tumor promotion by inhibitors of protein phosphatases 1 and 2A : the okadaic acid class of compounds. Adv Cancer Res. 1993 ; 61 : 143-194 https://doi.org/10.1016/S0065-230X(08)60958-6
  7. Kiguchi K, Glesne D, Chubb CH, Fujiki H, Huberman E : Differential induction of apoptosis in human breast tumor cells by okadaic acid and related inhibitors of protein phosphatases 1 and 2A. Cell Growth Differ. 1994 ; 5 : 995-1004
  8. Ishida Y, Furukawa Y, Decaprio JA, Saito M, Griffin JD : Treatment of myeloid leukemic cells with the phosphatase inhibitor okadaic acid induces cell cycle arrest at either G1/S or G2/M dependingon dose. J Cell Physiol. 1992 ; 150 : 484-492 https://doi.org/10.1002/jcp.1041500308
  9. Kiguchi K, Giometti C, Chubb CH, Fujiki H, Huberman E : Differentiation inductionin human breast tumor cells by okadaic acid and related inhibitors of protein phosphatases 1 and 2A. Biochem Biophys Res Commun. 1992 ; 189 : 1261-1267 https://doi.org/10.1016/0006-291X(92)90209-4
  10. Giese G, Wiegers W, Kubbies M, Scherbarth A, Traub P : Okadaic acid Co-induces vimentin expression and cell cycle arrest in MPC-11 mouse plasmacytoma cells. J Cell Physiol. 1995 ; 163 : 145-154 https://doi.org/10.1002/jcp.1041630117
  11. Morimoto Y, Morimoto H, Okamura H, et al : Upregulation of the expressionofFas antigenand Fas ligand in a human submandibular gland ductal cell line by okadaicacid. Arch Oral Biol. 2000 ; 45 : 657-666 https://doi.org/10.1016/S0003-9969(00)00038-8
  12. Suganuma M, Fujiki H, Suguri H, et al : Okadaic acid : an additional non-phorbol-12-tetradecanoate-13-acetate-type tumor promoter. Proc Natl Acad Sci USA. 1988 ; 85 : 1768-1771 https://doi.org/10.1073/pnas.85.6.1768
  13. Li DW, Xiang H, Mao YW, et al : Caspase-3 is actively involved in okadaic acid-induced lens epithelial cell apoptosis. Exp Cell Res. 2001 ; 266 : 279-291 https://doi.org/10.1006/excr.2001.5223
  14. Kwon TK : Phorbol myristate acetate inhibits okadaic acid-induced apoptosis and downregulation of X-linked inhibitor of apoptosis in U937 cells. Biochem Biophys Res Commun. 2001 ; 287 : 135-141 https://doi.org/10.1006/bbrc.2001.5531
  15. Grutter MG : Caspases : key players in programmedcell death. Curr Opin Struct Biol. 2000 ; 10 : 649-655 https://doi.org/10.1016/S0959-440X(00)00146-9
  16. Deveraux QL, Leo E, Stennicke HR, Welsh K, Salvesen GS, Reed JC : Cleavage of human inhibitor of apoptosis protein XIAP resultsinfragments with distinct specificities for caspases. EMBO J. 1999; 18 : 5242-5251 https://doi.org/10.1093/emboj/18.19.5242
  17. An B, Dou QP : Cleavage of retinoblastoma protein during apoptosis : an interleukin 1 beta-converting enzyme-like protease as candidate. Cancer Res. 1996 ; 56 : 438-442
  18. Koriyama H, Kouchi Z, Umeda T, et al : Proteolytic activation of protein kinase C delta and epsilonby caspase-3 in U937 cells during chemotherapeutic agent-induced apoptosis. Cell Signal. 1999 ; 11 : 831-888 https://doi.org/10.1016/S0898-6568(99)00055-8
  19. Park JW, Choi YJ, Suh SI, et al : Bcl-2 overexpression attenuates resveratrol-induced apoptosis in U937 cells by inhibition of caspase-3 activity. Carcinogenesis. 2001 ; 22 : 1633-1639 https://doi.org/10.1093/carcin/22.10.1633
  20. Decker P, Isenberg D, Muller S : Inhibition of caspase-3-mediated poly (ADP-ribose) polymerase(PARP) apoptotic cleavage by human PARP autoantibodies and effect on cells undergoing apoptosis. J Biol Chem. 2000 ; 275 : 9043-9046 https://doi.org/10.1074/jbc.275.12.9043
  21. Qi XM, He H, Zhong H, Distelhorst CW : Baculovirus p35 and Z-VAD-fmk inhibit thapsigargin-induced apoptosis of breast cancer cells. Oncogene. 1997 ; 15 : 1207-1212 https://doi.org/10.1038/sj.onc.1201290
  22. Deveraux QL, Stennicke HR, Salvesen GS, Reed JC : Endogenous inhibitors of caspases. J Clin Immunol. 1999 ; 19 : 388-398 https://doi.org/10.1023/A:1020502800208
  23. Roy N, Deveraux QL, Takahashi R, Salvesen GS, Reed JC : The c-lAP-1 and c-lAP-2 proteins are direct inhibitors of specific caspases. EMBO J. 1997 ; 16 : 6914-6925 https://doi.org/10.1093/emboj/16.23.6914
  24. Li P, Nijhawan D, Budihardjo I, et al : Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 1997 ; 91 : 479-489 https://doi.org/10.1016/S0092-8674(00)80434-1
  25. Yang J, Liu X, Bhalla K, et al : Prevention of apoptosis by Bcl- 2 : release of cytochrome c from mitochondria blocked. Science. 1997 ; 275 : 1129-1132 https://doi.org/10.1126/science.275.5303.1129
  26. Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer DD : The release of cytochrome c from mitochondria : a primary site for Bcl-2 regulation of apoptosis. Science. 1997 ; 275 : 1132-1326 https://doi.org/10.1126/science.275.5303.1132